Project Summary The major problem in cancer treatment is that, although patients respond to the initial treatment with cancer remission, the vast majority of the treated patients will have tumor recurrence in the primary and metastatic sites. For patients receiving immunotherapy, the recurring tumor cells frequently become antigen-loss-variants (ALVs) due to pressure and selection by the immune system. ALV outgrowth is an important mechanism by which tumors survive the immune attack and render immunotherapies targeting a single or multiple antigens ineffective. Recently, we discovered that when primed under Th9-polarizing conditions, nave CD8+ T cells could also differentiate into an IL-9-producing Tc9 subset (Lu et al, Proc Natl Acad Sci USA, 2014). Although less cytolytic in vitro as compared with Tc1 cells, adoptive transfer of tumor-specific Tc9 cells elicits a significantly greater antitumor response against large established melanoma (B16 and B16-OVA) and colon (MC38-gp100) tumors. More importantly, our preliminary studies showed that adoptively transferred tumor (OVA)-specific Tc9 but not Tc1 cells eradicated not only OVA-expressing tumor cells but also large established chimeric tumors containing both OVA-expressing and OVA-negative tumor cells, as well as OVA-negative tumor cells grown on the contralateral flank of mice, indicating that the Tc9 cells could mediate the killing of local bystander and remote ALVs in vivo. Based on these novel findings, we hypothesize that the Tc9 subset may be a superb effector T-cell subset for cancer immunotherapy to eradicate primary and recurrent ALV tumors. Aim 1 will determine whether and how Tc9 cells mediate killing of ALVs via epitope spreading and induction of a host CTL response against other antigens expressed by the tumor cells, and Aim 2 will determine the potential of human tumor-specific Tc9 cells in killing human primary and ALV tumors in vivo. These innovative and mechanistic studies will shed light on the mechanisms underlying Tc9 cell-mediated antitumor immunity and will thus establish a foundation for translating this discovery into more effective immunotherapies using tumor-specific T-cell subsets in human cancers.